Systematics and Biodiversity

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Systematic consideration of floral microcharacters of the South American Chrysolaena (, )

Gisela M. Via do pico, Alvaro J. Vega & Massimiliano Dematteis

To cite this article: Gisela M. Via do pico, Alvaro J. Vega & Massimiliano Dematteis (2016) Systematic consideration of floral microcharacters of the South American genus Chrysolaena (Vernonieae, Asteraceae), Systematics and Biodiversity, 14:2, 224-243, DOI: 10.1080/14772000.2015.1134699

To link to this article: http://dx.doi.org/10.1080/14772000.2015.1134699

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Download by: [181.1.176.133] Date: 30 January 2016, At: 07:22 Systematics and Biodiversity (2016), 14(2): 224À243

Systematic consideration of floral microcharacters of the South American genus Chrysolaena (Vernonieae, Asteraceae)

GISELA M. VIA DO PICO, ALVARO J. VEGA & MASSIMILIANO DEMATTEIS Instituto de Botanica del Nordeste (UNNE-CONICET), Casilla de Correo 209, CP 3400 Corrientes, Argentina (Received 11 August 2015; accepted 3 November 2015)

Floral microcharacters of the genus Chrysolaena H. Rob. (Vernonieae, Asteraceae) were analysed in detail for the first time in order to evaluate the taxonomic position of conflictive species in the group. The results were also compared with studies carried out in species of related genera. In addition to distinctive microcharacters previously studied in some species of the genus, other characters such as trichome types of the corolla, style, anthers and cypselae have been analysed for the first time. The presence of glandular apical appendage and cypselae are common characteristics among species Chrysolaena.In addition to these, this study shows that presence of glands on the style and corollas is another consistent characteristic in the genus. However, the absence of basal stylar node would not be a diagnostic character since this varies widely among species. The results indicate that most of the microcharacters of Chrysolaena analysed are quite consistent in the genus, but they are no more consistent than the pollen morphology (type ’C’) and chromosome base number (x D 10). Until now, these last two features would be most useful for separating Chrysolaena from the related genera and . At species level, the results show that related species can be distinguished by the different combinations of floral microcharacters. The value of microcharacters could be increased if they are combined with other morphological, cytological, and palynological data. Keywords: anthers, crystals, cypselae, idioblasts, Lepidaploinae, style, , trichomes

Introduction With the emergence of ’The new synantherology’ (King & Robinson, 1970), dozens of new genera have The Asteraceae comprise between 1600À2000 genera been split out of several traditional genera of Asteraceae and 24,000À30,000 species divided into 13 subfamilies on the basis of micromorphological characters. In subse- and43tribes(Paneroetal.,2014). The family has pre- quent years, several authors have used a cluster of micro- sented continuing taxonomic problems as a result of its morphological characters in taxonomic studies in large size and its recurring combinations of superficial char- groups as Senecioneae, , Vernonieae, Eupator- acters (Robinson, 1999). The tribe Vernonieae is a clear ieae, Inuleae (Dematteis, 2009, Faust & Jones, 1973; Isa-

Downloaded by [181.1.176.133] at 07:22 30 January 2016 example of one of the most complex and less elucidated wumi, 1999; Isawumi, El-Ghazaly, & Nordenstam, 1996; groups. It is one of the largest within the Asteraceae Liu & Yang, 2011; Narayana, 1979; Pruski, 2012; Riva, with approximately 1500À1600 species distributed in Pozner, & Freire, 2009; Robinson, 1988a, 1988b, 1992, America, Asia, and Africa (Keeley, Forsman, & Chan, 1999). Some uses of microcharacters are, for example, 2007; Keeley & Robinson, 2009). Good taxonomic species identification from small fragments of the , understanding of Vernonieae has been difficult to or the relocation or change of taxonomic position of taxa achieve, especially among certain New World taxa (Robinson, 2009). (Ekman, 1914; Gleason, 1906;Jones,1979, 1981; The taxonomic history of the tribe Vernonieae has Keeley & Jones, 1977). The source of much of this dif- changed over time and the huge or core genus ficulty lies with the extensive variability inherent in the Less. was suffering divisions, fragmentations, and rear- group, the occurrence of gradual morphologies and rangements. Based on morphological, phytochemical, overlapping characters with numerous combinations of cytological, palynological, and molecular studies a total autoapomorphic states (Keeley & Jones, 1977;Keeley of 126 genera distributed in 21 subtribes, 15 in the New &Turner,1990; Robinson, 1999). World, and six in the Old World are recognized for the tribe (Keeley & Robinson, 2009). Most of the emerged Correspondence to: Gisela M. Via do Pico. E-mail: gisela_ and resurrected new genera were established based on [email protected]

ISSN 1477-2000 print / 1478-0933 online Ó The Trustees of the Natural History Museum, London 2016. All Rights Reserved. http://dx.doi.org/10.1080/14772000.2015.1134699 Floral microcharacters of Chrysolaena (Asteraceae) 225

micromorphological characters. In taxonomic studies on conflictive species of the genus; and (3) determine the sys- American Vernonieae, Robinson (1999) attached great tematic value of these microcharacters in specific segrega- importance to the value of floral microcharacters for sub- tion of the taxa. tribal and generic delimitations (e.g., the style base, the anther appendages, the carpopodium shape, and the shape of cypsela wall crystals). Some of these characters were Materials and methods used as major distinguishing features in the segregation of genera such as Chrysolaena H. Rob., Lessingianthus H. The specimens for this study were obtained from living Rob., Vernonanthura H. Rob., and Lepidaploa H. Rob., material and the herbarium of Instituto de Botanica del previously members of the core genus Vernonia Nordeste (CTES). A complete voucher list is given in the (Robinson, 1988a, 1988b, 1990, 1999). Other characters Appendix. taken into account were the morphology of the pollen For the micromorphological observations with a light grains, chromosome number, chemical composition, and microscope (LM) , florets and fruits were removed from inflorescence shape (Dematteis, 2007, 2009; Robinson, herbarium specimens. Samples were rehydrated in 2009). warm soapy water and dissected under a stereoscopic Chrysolaena belongs to the subtribe Lepidaploinae S. microscope. They were then mounted in Hoyer’s C. Keeley & H. Rob. and comprises 18 species geographi- solution (Anderson, 1954; King & Robinson, 1970). cally concentrated in the central region of South America, Observations were done with a LM equipped with a dig- with the main center of diversity in southern Brazil and ital photographic camera (Canon Power Shot A 640). northeast of Argentina (Dematteis, 2009; Robinson, Trichome types were drawn with a camera lucida 1988b). Most species have a wide geographic distribution, microscope. although there are some endemic entities. The species of Observations with a scanning electron microscope genus are perennial herbs or erect shrubs with well devel- (SEM)were performed on herbarium and living material. oped xylopodia. They are characterized by sericeous or Herbarium samples were rehydrated with warm soapy velutine indumentum, mainly in stems, leaves and phylla- water and fixed with FAA. Disc florets were dehydrated ries, style without basal node, and glandular anthers and in a graded series of acetone and critical point-dried with cypselae (Robinson, 1988b). However these features are liquid CO2. Dried samples were partially dissected, only known for seven species: C. flexuosa (Sims) H. Rob., mounted on stubs, coated with gold/palladium and C. lithospermifolia (Hieron.) H. Rob., C. obovata (Less.) observed with SEM (JEOL 5800 LV). Dematt., C. oligophylla (Vell.) H. Rob., C. platensis For the description of microcharacters, terminology (Spreng.) H. Rob., C. propinqua (Hieron.) H. Rob., and C. proposed by Metcalfe and Chalk (1979), Narayana verbascifolia (Less.) H. Rob. (Robinson, 1988b). Other (1979), Ramayya (1962), and Robinson (1988a, 1988b, distinctive characteristics of the genus are the presence of 1990, 1999) was used. pollen grain type ’C’ (tricolporate, echinolophate with 29 lacunae) and the base chromosome number x D 10 (Dematteis, 2009; Oliveira, Forni-Martins, & Semir, Statistical analyses 2007a, 2007b; Oliveira, Semir, & Forni-Martins, 2012; A data matrix with 59 OTUs £ 13 characters was made. Downloaded by [181.1.176.133] at 07:22 30 January 2016 Via do Pico & Dematteis, 2012, 2013a, 2013b, 2014). The size, the presence of idioblasts and crystals on the Chrysolaena is closely related to Lessingianthus and fruit were not considered because these showed great Lepidaploa, with which it shares some characteristics. intraspecific variation. Also taxa with incomplete infor- With Lepidaploa, for example, it shares the presence of mation were not considered. The characters were coded glands in anthers and cypselae, elongated crystals of in binary (Table S1, see online supplemental material, cypsela wall, and pollen grains type ’C’. However, the which is available from the article’s Taylor & Francis last feature is not exclusive to Lepidaploa, since pollen Online page at http://dx.doi.org/10.1080/14772000. type ’D’ and ’G’ can also occur. By contrast, with Lessin- 2015.1134699). The homogeneous characters among all gianthus it shares the absence of basal style node (Demat- species were not considered as variables, and nor were the teis, 2007, 2009; Robinson, 1990). Recent molecular presence of idioblasts and calcium oxalate crystals, since studies suggest that Chrysolaena is more closely related they showed high intraspecific variation. A cluster analy- to Lepidaploa, with Lessingianthus as sister group sis UPGMA (Unweighted Pair Group Method with Arith- (Keeley et al., 2007). metic Mean) and a principal coordinate analysis (PCoA) Based on this background, the aims of this study are to: using Dice distance index (Nei & Li, 1979) were per- (1) characterize floral microcharacters of the genus Chrys- formed. The program InfoStat 2012 version was used for olaena; (2) evaluate the taxonomic position of the both analyses (Di Rienzo et al., 2012). 226 G. M. Via do Pico et al.

Results corolla tube (in a few species) were observed. We found Micromorphological data of all the Chrysolaena species four types of trichomes based on the glandular or non- are presented in Tables 1 and 2. glandular nature and cellular structure, as follows. Type I: simple filiform trichome (Figs 2, 17, 20, 40): non-glandular, unicellular formed by a single elongated Trichomes cylindrical cell of acute apex; or multicellular with a foot Glandular and non-glandular trichomes on the anther formed by 1 or 2 basal cells and a cylindrical elongated appendages, the corolla lobes, the cypselae, and the terminal cell of acute apex. The side walls are thin or

Table 1. Qualitative and quantitative data of the floral microcharacters of Chrysolaena. The “x” indicates the presence of character. The dash indicates missing or immature material.

Trichomes type of corolla Anthers Style

Apical Trichome appendage typesGlandular Basal node Species and trichomes voucher herbarium I II IIIA IIIB IV Shape Size (mm) IIIA I Shape (Type IIIA) Developed Ring of cells

C. campestris 8707 x x x ovate 0.6À0.8 x x sagittate x x C. candelabrum 45761 x x lanceolate 0.7À0.8 x sagittate x x C. cognata 46948 x x ovate 0.6À0.8 x cleft x x C. cognata 3399 x x ovate 0.5À0.7 x cleft x C. cognata 3053 x ovate 0.5À0.6 x cleft x C. cognata 6 x ovate 0.6À0.8 x sagittate x C. cognata 3040 x x ovate 0.5À0.6 x sagittate x C. cordifolia 24905 x x lanceolate 1.0À1.1 x x sagittate x C. cordifolia 3975 x lanceolate 0.9À1.0 x sagittate x C. cristobaliana 882 x ovate-lanceolate 0.4À0.5 x sagittate x x C. cristobaliana 34544 x ovate-lanceolate 0.4À0.6 x sagittate x x C. cristobaliana 41 x ovate-lanceolate 0.5À0.6 x sagittate x x C. desertorum 23783 ------C. desertorum 5840 x ovate 0.4À0.5 x cleft x C. desertorum 35640 x x ovate 0.5À0.6 x sagittate x C. desertorum 24369 x ovate 0.3À0.6 x sagittate x x C. dusenii 4688 x x ovate 0.7À1.0 x sagittate x x C. dusenii 2093 x lanceolate 0.8À1.0 x sagittate x x

Downloaded by [181.1.176.133] at 07:22 30 January 2016 C. dusenii 1286 x lanceolate 0.7À0.9 x sagittate x x C. dusenii 12012 x lanceolate 0.7À1.0 x sagittate x x C. flexuosa 45 x ovate-lanceolate 0.7À0.8 x x cleft x C. flexuosa 5 x ovate 0.5À0.7 x sagittate x C. flexuosa 79091 x x ovate 0.6À0.7 x cleft x C. flexuosa 44199 x ovate 0.6À0.7 x sagittate x x C. guaranitica 12524 x orbicular 0.5À0.7 sagittate C. lithospermifolia 9 x ovate 0.5À0.6 x cleft x C. lithospermifolia 15 x ovate 0.5À0.6 x cleft x x C. lithospermifolia 42055 x ovate-lanceolate 0.5À0.6 x cleft x C. lithospermifolia 5884 x ovate 0.7À0.8 x sagittate x C. nicolackii 5533 x x lanceolate 0.8À0.9 x sagittate x x C. nicolackii 12023 x x ovate-lanceolate 0.4À0.7 x cleft x x C. nicolackii 6214 x x lanceolate 0.9À1.1 x sagittate x C. nicolackii 682 x x ovate-lanceolate 0.8À0.9 x sagittate x x C. obovata 3475 x x x ovate-lanceolate 0.4À0.6 sagittate x C. obovata 1736 x lanceolate 0.5À0.6 x sagittate x x

(continued) Floral microcharacters of Chrysolaena (Asteraceae) 227

Table 1. (Continued )

Trichomes type of corolla Anthers Style

Apical Trichome appendage typesGlandular Basal node Species and trichomes voucher herbarium I II IIIA IIIB IV Shape Size (mm) IIIA I Shape (Type IIIA) Developed Ring of cells

C. obovata 36 x lanceolate 0.4À0.6 sagittate x x C. obovata 17361 x lanceolate 0.4À0.5 x sagittate x x C. oligophylla 270 x ovate 0.8À0.9 x sagittate x C. oligophylla 1729 x ovate 0.5À0.7 x sagittate x C. oligophylla 5455 x ovate 0.4À0.6 x sagittate x C. oligophylla 2953 x x x ovate 0.5À0.7 x sagittate x x C. platensis 3083 x lanceolate 0.6À1.0 x sagittate x C. platensis 5123 x lanceolate 1.0À1.1 x sagittate x x C. platensis 609 x lanceolate 0.7À0.8 x sagittate x C. platensis 1478 x x lanceolate 0.7À0.8 x sagittate x C. propinqua 913 x x ovate 0.6À0.8 x cleft x C. propinqua 3038 x x ovate 0.6À0.7 x sagittate x x C. propinqua 20 x x ovate 0.4À0.6 x sagittate x x C. propinqua 25 x x ovate 0.3À0.5 x sagittate x C. propinqua 35815 x x ovate 0.3À0.4 x sagittate x x C. propinqua D y O x x ovate 0.5À0.6 x sagittate x x C. sceptrum 76914 x x ovate 0.7À0.8 x sagittate x C. sceptrum 30442 x x ovate 0.6À0.7 x sagittate x C. sceptrum 3307 x x ovate 0.5À0.7 x sagittate x C. simplex 547 x x orbicular 0.6À0.7 sagittate C. simplex 5704 x x orbicular 0.5À0.7 sagittate C. simplex 69630 x x x orbicular 0.7À0.8 sagittate C. simplex 23722 x x orbicular 0.6À0.7 sagittate C. simplex 6057 x x x orbicular 0.6À0.7 sagittate C. verbascifolia 12031 x ovate-lanceolate 0.5À0.6 x sagittate x C. verbascifolia 34 x x x lanceolate 0.7À0.9 x sagittate x x

slightly thickened. Trichome type II differs from papillae of thin walls in each row. It has a cuticular vesicle in the Downloaded by [181.1.176.133] at 07:22 30 January 2016 in that the latter are short and rounded emergences of the terminal apex that in frontal view usually presents bilobed membrane of epidermal cells. appearance. This may persist or collapse. Type IIIB Type II: twin hair (Figs 13, 18, 21, 41): non-glandu- (Figs 3, 11, 43): simple or compound foot, biseriate body lar, multicellular, biseriate, formed by four cells, two formed by 4À7 cells on each row, differentiated in stalk basal cells and two ’hair cells’, cylindrical, sharp ends and head. The basal cells of the rows are generally longer and thick walls; arranged parallel to each other. The latter and of different size; towards the apex are of smaller size may have the same length as each other, or one may be and homogeneous with each other. The cell walls are thin. longer; they may be completely joined together or sepa- The cuticular vesicle can be persistent or collapse. rate at the ends or at the base. Trichomes type II of the Type IV: long-stalked glandular trichome (Figs 12, anther appendages and the corollas usually have small 23, 44): foot formed by 1 or more cells. The body is biseri- variations of the typical morphology of twins hairs pres- ate, elongated and differentiated into stalk and head. The ent on fruits. They may have from 3 to 8 cells arranged in stalk is formed by 3-4 long cells in each row, more or less two rows, with the two terminal cells cylindrical and with uniform in width. Cells of 2 rows are arranged alternately sharp ends. These atypical morphologies were observed and can be of different sizes. The head is formed by 1À2 in few specimens. cells on each row, sharply demarcated with the stalk, or Type III: short-stalked capitate glands: Type IIIA continuous. The cells have thin walls or slightly thickened (Figs 1, 10, 19, 29, 30, 42): short trichomes; narrow foot in certain parts. In the early stages of development are formed by 1 or 2 cells. The biseriate body has 2À4 cells seen as glandular biseriate trichomes. 228 G. M. Via do Pico et al.

Table 2. Qualitative and quantitative data of cypselae of Chrysolaena. The “x” indicates the presence of character.

Crystals Trichomes Species and voucher Shape Size (mm) type IIIA Idioblasts Cubic Estiloid Prismatic Druses

C. campestris 8708 turbinate 1.7À1.9 x x x x C. candelabrum 45761 turbinate 1.6À1.7 x x x x x C. cordifolia 24905 turbinate 1.3À1.5 x x x C. cordifolia 3975 turbinate 1.5À1.7 x x C. cognata 46948 cylindrical-turbinate 2.9À3.2 x x x C. cognata 3399 cylindrical-turbinate 1.9À2.0 x x x x x C. cognata 3053 cylindrical-turbinate 2.5À2.8 x x x C. cognata 6 cylindrical-turbinate 1.6À1.9 x x x x C. cognata 3040 cylindrical-turbinate 2.4À2.6 x x x x C. cristobaliana 882 turbinate 1.7À2.1 x x x x x C. cristobaliana 34544 turbinate 2.0À2.3 x x C. cristobaliana 41 turbinate 2.0À2.2 x x x C. desertorum 23783 cylindrical-turbinate 2.9À3.1 x x x C. desertorum 35640 cylindrical-turbinate 2.0À2.1 x x x C. desertorum 24369 cylindrical-turbinate 1.9À2.0 x x C. desertorum 5840 cylindrical-turbinate 2.3À2.5 x C. dusenii 4688 turbinate 2.2À2.9 x x x C. dusenii 2093 turbinate 2.9À3.1 x x x C. dusenii 12012 ------C. dusenii 1286 turbinate 2.0À2.4 x x x x C. flexuosa 45 cylindrical-turbinate 2.0À2.3 x x x x C. flexuosa 5 cylindrical-turbinate 2.0À2.2 x x x C. flexuosa 79091 turbinate 2.0À2.1 x x x x C. flexuosa 44199 turbinate 2.0À2.2 x x x x C. guaranitica 12524 turbinate 2.0À2.3 x x x x C. lithospermifolia 9 turbinate 2.0À2.1 x x x x C. lithospermifolia 15 turbinate 1.8À2.0 x x x x C. lithospermifolia 42055 turbinate 2.0À2.3 x x x x x C. lithoseprmifolia 5884 turbinate 2.0À2.3 x x x x C. nicolackii 5533 cylindrical-turbinate 1.8À2.0 x x x x C. nicolackii 12023 cylindrical-turbinate 2.6À2.9 x x C. nicolackii 6214 cylindrical-turbinate 1.7À2.0 x x x x À Downloaded by [181.1.176.133] at 07:22 30 January 2016 C. nicolackii 682 cylindrical-turbinate 2.0 2.2 x x x C. oligophylla 270 turbinate 2.0À2.2 x x x C. oligophylla 1729 turbinate 1.7À1.8 x x C. oligophylla 5455 turbinate 2.0À2.2 x x x x x C. oligophylla 2953 turbinate 1.8À2.0 x x x C. obovata 3475 cylindrical-turbinate 1.9À2.1 x x x C. obovata 1736 cylindrical-turbinate 1.8À2.0 x x x x C. obovata 36 cylindrical-turbinate 2.3À2.5 x x x C. obovata 17361 cylindrical-turbinate 2.0À2.2 x x x C. platensis 3083 turbinate 2.5À2.7 x x x x x C. platensis 5123 turbinate 1.9À2.1 x x x x C. platensis 609 turbinate 2.2À2.5 x x x x C. platensis 1478 turbinate 2.0À2.5 x x x x C. propinqua 913 turbinate 2.0À2.2 x x x x C. propinqua 3038 turbinate 1.7À2.0 x x x C. propinqua 20 turbinate 2.0À2.1 x x x C. propinqua 25 turbinate 2.0À2.2 x x x C. propinqua 35815 turbinate 2.0À2.3 x x x x

(continued) Floral microcharacters of Chrysolaena (Asteraceae) 229

Table 2. (Continued )

Crystals Trichomes Species and voucher Shape Size (mm) type IIIA Idioblasts Cubic Estiloid Prismatic Druses

C propinqua D y O turbinate 2.0À2.2 x x x x C. sceptrum 76914 turbinate 2.0À2.3 x x x x C. sceptrum 30442 turbinate 2.7À2.9 x x x x x C. sceptrum 3307 turbinate 2.0À2.2 x x x x C. simplex 547 turbinate 2.9À3.0 x x C. simplex 5704 turbinate 2.5À2.7 x x x C. simplex 69630 turbinate 2.2À2.5 x C. simplex 23722 cylindrical-turbinate 1.9À2.0 x x C. simplex 6057 cylindrical-turbinate 2.0À2.3 x x C. verbascifolia 12031 cylindrical-turbinate 2.0À2.3 x x C. verbascifolia 34 cylindrical-turbinate 2.0À2.2 x x

Corolla pubescence Base of anther thecae and apical appendages All the species have papillae on the adaxial surface of Chrysolaena has sagittate anthers. A smaller number of corolla lobes. Most of the species have glandular tri- entities have cleft anthers. Regarding to the insertion the chomes type IIIB (Figs 1, 16, 18), usually accompanied anthers are calcarate, as the thecae extend below the point by type I (Fig. 20) or twin hairs (Figs 18 and 21). Chryso- where the filament connects with the anther. laena simplex (Less.) Dematt. and C. guaranitica Dematt., Apical anther appendages are flat and show variable have only non-glandular trichomes type IA on corolla shapes: ovate, ovate-lanceolate, lanceolate, and orbicular lobes (Figs 2 and 17). Chrysolaena nicolackii H.Rob. and (Fig. 8, 9, 26, 27, 45À55). In some species, they are not the population of C. verbascifolia No. 34 have trichomes constricted at the base and are the same width as the the- type IIIB on the corolla tube and corolla lobes, respec- cae. The size varies from 0.4 to 1.1 mm long. In most spe- tively (Fig. 3). Chrysolaena simplex has trichomes type cies the appendages are ovate to ovate-lanceolate with IV on the corolla tube; this feature is unique to this species glandular trichomes type IIIA (Fig. 26); except C. sim- among all of those examined in this study (Figs 4, 22, 23). plex, C. obovata, and C. guaranitica, where they are No species exhibit entirely glabrous corolla. All trichomes orbicular and glabrous (Figs 9 and 27), and in two popula- described are in corollas of Chrysolaena. tions of C. obovata Nos 3475 and 36 where appendages are glabrous. Chrysolaena campestris (DC.) Dematt., C. cordifolia Dematt. No. 24905, and C. flexuosa No. 45 Style show trichomes type IA in addition to type IIIA.

Downloaded by [181.1.176.133] at 07:22 30 January 2016 Chrysolaena has style type ’vernonioid’: bifid, slender, with sweeping hairs along the branches as well on the upper part of the shaft. It has stigmatic papillae on the Cypselae and pappus inner top of the branches, and is glabrous to sub-glabrous The fruits of Chrysolaena species are very similar in towards the base. Non-glandular (type I) and glandular tri- shape and indumentum (see Table 2). The cypselae are chomes (IIIA) occur from the middle part to the apex turbinate (most species) or cylindrical-turbinate (Figs 7 and 24). In three species, C. simplex, C. obovata, (Fig. 56À65). The size varies from 1.3 mm long in C. cor- and C. guaranitica, trichomes type IIIA are absent difolia No. 24905 to 3.2 mm long in C. cognata (Less.) (Fig. 27). Dematt. No. 46948. Basic colour is brown; however there is variation from dark brown to yellowish brown. In all taxa examined non-glandular trichomes type II (twin Basal stylar node hairs) occur throughout the surface of fruit, and most also Most specimens have style without basal nodule (Fig. 6). have glandular trichomes type IIIA (Figs 13, 28À30, 33, However, in some entities a conspicuous group of sclerifi- 34, 38), located mainly at the base, linearly or scattered. cate cells was observed (Fig. 5) and in others, a little Three species exhibit no glandular cypselae: C. guarani- developed nodule formed by a few rows of cells (Fig. 25). tica, C. simplex, and C. obovata Nos 17361, 36 (Fig. 37). This character varied not only between species but also Besides trichomes, the cypselae usually possess idio- between populations of the same species (see Table 1). blasts. These are more or less rounded and conspicuous 230 G. M. Via do Pico et al. Downloaded by [181.1.176.133] at 07:22 30 January 2016

Fig. 1À15. Floral microcharacters of Chrysolaena (LM). (1) C. cordifolia No. 24905: corolla lobes with glandular trichomes type IIIA; (2) C. guaranitica: corolla lobes with non-glandular trichomes type I; (3) C. nicolackii No. 12023: corolla tube with trichomes type IIIB; (4) C. simplex: corolla tube with trichomes type IV; (5) C. campestris No. 8707: basal stylar node; (6) C. lithospermifolia No. 5884: style with- out basal node; (7) C. candelabrum No. 45761: style branch with trichomes type IIIA; (8) C. platensis No. 3083: lanceolate apical anther appendage with trichomes type IIIA; (9) C. simplex No. 5704: orbicular and glabrous apical anther appendage; (10) C. candelabrum No. 45761: glandular trichome type IIIA of style; (11) C. nicolackii No. 5533: trichome type IIIB; (12) C. simplex:trichometypeIV;(13)C. nicolackii N 682: trichomes type II and IIIA, and crystals of the cypsela; (14) C. lithospermifolia No. 8: detail of carpopodium; (15) C. cognata No. 3053: idioblasts on the cypsela wall. Scale Bars:50mmin(1À8, 12, 14); 100 mmin(9À11, 13, 15). Floral microcharacters of Chrysolaena (Asteraceae) 231 Downloaded by [181.1.176.133] at 07:22 30 January 2016

Fig. 16À27. Floral microcharacters of Chrysolaena (SEM). (16) C. candelabrum: corolla lobes with trichomes type I and IIIA; (17) C. guaranitica: corolla lobe with trichomes type I; (18) C. oligophylla No. 2953: corolla lobe with trichomes type II and IIIA; (19) C. pla- tensis No. 1478: detail of trichomes type IIIA of corolla; (20) C. sceptrum No. 3307: detail of trichomes type I of corolla; (21) C. obovata No. 3475: trichomes type II (twin hairs) of corolla; (22À23) C. simplex: corolla tube and detail of trichome type IV, respectively; (24) C. cristobaliana: detail of style branch with trichome type IIIA; (25) C. verbascifolia No. 34: basal stylar node poorly developed; (26) C. propinqua: trichomes type IIIA on the apical anther appendage; (27) C. simplex: style branches and anther appendages without glandular trichomes. 232 G. M. Via do Pico et al. Downloaded by [181.1.176.133] at 07:22 30 January 2016

Fig. 28À39. Floral microcharacters of Chrysolaena (SEM). (28À29) C. cognata No. 3053: (28) general view of cypsela; (29) cypsela wall with trichomes type II, IIIA and idioblasts; (30) C. flexuosa No. 5: trichomes type II and IIIA of the cypsela; (31) C. obovata No. 36: detail of cypsela wall; (32) C. propinqua No. 25: general view of cypsela; (33À35) C. sceptrum No. 3307: (33) detail of carpopodium with sub-squares cells; (34) trichomes of the cypsela wall; (35) external series bristle of pappus (flat bristles); (36) C. sceptrum No. 76914: general view of cypsela; (37) C. simplex No. 6057: general view of cypsela without glands; (38À39) C. verbascifolia No. 34: (38) detail of a portion of carpopodium, glandular trichomes and twin hairs; (39) detail of the cylindrical inner series bristles of pappus. Black arrows show the idioblasts. Floral microcharacters of Chrysolaena (Asteraceae) 233

Fig. 45À55. Anthers of Chrysolaena. (45) C. guaranitica No. 12524; (46) C. simplex No. 5704; (47) C. dusenii No. 1286; (48) À C. candelabrum No. 45761; (49) C. platensis No. 5123; (50) C. Fig. 40 44. Trichome types of florets and cypselae of Chryso- obovata No. 1736; (51) C. lithospermifolia No. 5884; (52) C. laena. (40) Type I: simple filiform trichome; (41) Type II: twin flexuosa No. 45; (53) C. desertorum No. 5840; (54) C. verbasci- hair, regular and atypical forms; (42) Type IIIA: short-stalked folia No. 12031; (55) C. flexuosa No. 44199. capitate glands. (43) Type IIIB; (44) Type IV: long-stalked glan- dular trichome.

structures, different from other cells that form the wall of Ovary wall crystals the fruit. With LM are clearly distinguishable by their Ovary walls present several forms of calcium oxalate crys- intense brownish colour. They are distributed irregularly À tals: cubic, prismatic, styloid, and druses. Their sizes and over the fruit wall and are arranged in groups of 2 8 cells À

Downloaded by [181.1.176.133] at 07:22 30 January 2016 density are variable between the specimens (Fig. 13 15). (Figs 15, 28, 29, 31). The presence of idioblasts also Most species showed crystals, but not always all the shapes. varies at intraspecific level. The most common crystals among species studied are pris- A biseriate and double pappus is present in the fruits of matic and cubic. Chrysolaena cristobaliana Dematt. Nos all species of Chrysolaena. The bristles have short teeth. 34544, 24369 and C. desertorum (Mart. ex DC.) Dematt. The external series is flat and shortest. The internal series No. 5840 do not exhibit crystals on the ovary walls. has cylindrical and longer bristles (Fig. 32, 35, 36, 39).

Carpopodium Cluster analysis (UPGMA) and principal The carpopodium is the basal abscission area of the cypsela coordinates analysis (PCoA) formed by one or more rows of cells which usually are mor- Figure 66 shows the phenogram of the UPGMA. Cluster phologically distinct from other cells of the wall cypselae. analysis divided the 59 OTUs into two main groups based In the taxa investigated the carpopodium is well developed on the shape of the apical anther appendage. Group 1 con- and differentiated; is cylindrical or slightly turbinate. It is tains species with orbicular apical appendages (Fig. 45 and ring-like without interruptions covering the cypsela base, 46), while in Group 2 are all other entities that may present and is formed by sub-oblong or square cells with thickened apical appendages ovate, lanceolate or ovate-lanceolate. walls (Figs 14, 33, 38). In a few species the carpopodium Group 1 is subdivided in two: group 1A, that contains taxa shows small crystals druses (e.g., C. nicolackii). with trichomes type IV on the corolla tube (C. simplex), and 234 G. M. Via do Pico et al. Downloaded by [181.1.176.133] at 07:22 30 January 2016

Fig. 56À65. Cypselae of Chrysolaena. (56) C. verbascifolia No. 34; (57) C. sceptrum No. 30442; (58) C. cognata No. 3053; (59) C. pla- tensis No. 5123; (60) C. obovata No. 36; (61) C. propinqua No. D y O s/n; (62) C. nicolackii No. 12023; (63) C. flexuosa No. 45; (64) C. flexuosa No. 44199; (65) C. cognata 30422. Turbinate cypselae in (56, 57, 64); Cylindrical-turbinate cypselae in (58À63, 65). Floral microcharacters of Chrysolaena (Asteraceae) 235

Fig. 66. Phenogram of 13 characters and 59 OTUs resulting from UPGMA based on the Dice coefficient.

group 1B, taxa with glabrous corolla tube (C. guaranitica). mainly on the presence of the different types of trichomes Subgroup 1A is divided, again, into two groups based on the on apical anther appendages and corollas. presence or absence of type II trichomes on the corolla, and The PCoA shows that 52.4% of the variation can be these groups are subdivided according to the fruit shape (tur- explained by the first two principal coordinates (PC), PC binate or cylindrical-turbinate). Group 2 includes the largest 1 and 2 (Fig. 67). PC 1 explains 35.5% of the total varia- number of taxa and is subdivided several times based tion, while PC 2 explains 16.9%. The PCoA diagram Downloaded by [181.1.176.133] at 07:22 30 January 2016

Fig. 67. Principal Coordinates Analysis (PCoA) of Chrysolaena species based on floral microcharacters. Abbreviations: PC À principal coordinate; camp À Chrysolaena campestris; cande À C. candelabrum; cog À C. cognata; cordi À C. cordifolia; cristo À C. critobali- ana; deser À C. desertorum; duse À C. dusenii; flex À C. flexuosa; guara À C. guaranitica; litho À C. lithospermifolia; nico À C. nico- lackii; obo À C. obovata; olig À C. oligophylla; plat À C. platensis; pro À C. propinqua; scep À C. sceptrum; sim À C. simplex; verb À C. verbascifolia. Circles show the taxon groups which correspond to the clustering phenogram of Fig. 66. 236 G. M. Via do Pico et al.

shows distinctive groups of taxa. These groups correspond form the hair and the degree of divergence of the hair cells to the grouping of phenogram of cluster analysis. at the apex. The trichome type IIIA correspond morphologically with the typical vesicular glands, usually observed as Discussion glandular dots in different organs of the Asteraceae. They usually contain sesquiterpene lactones (Ramayya, 1962; The species of Chrysolaena are completely uniform in the Robinson, 2009). This type of trichome is very common presence of papillae on the style, the insertion of the in Vernonieae and is the structure that Robinson (1988a, anther in the filament (calcarate), the pappus (biseriate 1988b, 1990) referred to as ’glands’ and noted in apical with external bristles flattened and short, and the inner anther appendages, the corolla lobes and the cypselae in cylindrical and long) and carpopodium (forming a cylin- his treatment of Lepidaploa complex. Previous studies drical or slightly turbinate ring, with oblong or sub-square revealed the occurrence of this type of glandular tri- cells). In addition to the microcharacters used by Robin- chomes on leaves, stems, and underground organs (rhizo- son (1988b) for circumscription of the genus, other char- phores) of Chrysolaena obovata, C. platensis, C. cognata, acters of taxonomic significance were analysed for the C. flexuosa, C. lithospermifolia, C. propinqua, and C. ver- first time. Below we discuss separately the microchar- bascifolia (Appezzato-da-Gloria et al., 2012; Sosa, Via do acters examined. Pico, & Dematteis, 2014). Similar morphology of trichome type IV was observed in petioles of Echinops echinatus Roxb. (Ramayya, Trichomes 1962), in leaves of Madia sativa Mol. (Asteraceae) (Carl- The Asteraceae family is characterized by glandular and quist, 1958) and members of the subtribe Espeletiinae in non-glandular trichomes (Metcalfe & Chalk, 1950, 1979) Heliantheae tribe (Robinson, 2009). These trichomes usu- in floral and vegetative organs, and even in the under- ally contain monoterpenes (Robinson, 2009). ground organs (Appezzato-da-Gloria, Hayashi, Cury, The use of trichomes is well established in comparative Soares, & Rochao, 2008; Appezzato-da-Gloria et al., systematic studies of several groups of angiosperms 2012). Type and proportion can vary throughout the plant because of their variety, wide occurrence, ease of prepara- and between different taxonomic levels (Werker, 2000). tion for study and close relation of their variation patterns Based on its glandular and non-glandular nature and the to the taxonomic system (Carlquist, 1961). Within Verno- number and organization of their cells, a total of five mor- nieae, numerous studies have supported the utility of tri- phological types of trichomes were described for Chryso- chomes for systematic studies (Faust, 1972; Hunter & laena. The morphologies observed are consistent with Austin, 1967; Urbatsch, 1972; Wagner et al., 2014). those described for other species of Asteraceae and Ver- The trichomes morphology in Chrysolaena is variable nonieae. According to these previous studies, the mor- and some types presented diagnostic value at species phologies of the trichomes here described as types I, II, level. The presence or absence of glandular trichomes is and IIIA are the most common among species (Angulo & the main character that allowed distinguishing some spe- Dematteis, 2014; Appezzato-da-Gloria et al., 2012; Freire, cies, because some morphotypes are restricted to certain Katinas, & Sancho, 2002; Isawumi, 1999, Isawumi et al., structures or to particular species. For example, C. simplex Downloaded by [181.1.176.133] at 07:22 30 January 2016 1996; Metcalke & Chalk, 1950; Narayana, 1979; Ram- and C. nicolackii can be separated from the other species ayya, 1962; Redonda-Martınez, Villasenor,~ & Terrazas, by the presence of glandular trichomes on corolla tube. 2012; Robinson, 2009; Sancho & Katinas, 2002). The Besides, C. simplex differs by the exclusive presence of morphology of trichomes type I agree with those glandular trichomes type IV. Moreover, C. guaranitica described as types I and II on the corollas of most Lessin- and C. simplex have only non-glandular trichomes type I gianthus species (Angulo & Dematteis, 2014). The type II on corolla lobes, and lack glandular trichomes type IIIA, trichomes are of the same morphology as the twin hairs, while the remaining species possess them. The lack of or also called double hairs or Zwillingshaare (Kraus, glandular trichomes on corolla lobes and cypselae are typ- 1866), typical of the fruits of most genera of Asteraceae ical of most species of Lessingianthus (Robinson, 1988a). (Hess, 1938; Ramayya, 1962; Robinson, 2009). However, variations of basic type were observed, mainly on corollas and anthers. Previous studies have shown that twin hairs may vary morphologically in the size, length, wall thick- Corolla pubescence ness, or the degree of divergence, the degree of develop- Pubescence of corolla is a feature widely used in Verno- ment and reduction of hair cells and/or basal cells (Freire nieae systematics (Isawumi, 1999). However, it has only & Katinas, 1995; Hess, 1938; Ml’khi R.ike & Norden- been analysed in detail in Old World genera such as Bac- stam, 2012). Atypical morphologies seen in Chrysolaena charoides Moench (Isawumi et al., 1996), Cyanthillium showed variations in the length, the number of cells that Blume (Isawumi, 1995b), and some African species of Floral microcharacters of Chrysolaena (Asteraceae) 237

Vernonia s.l. (Isawumi, 1995a). For American Vernonieae In this study the trichome types of the style were ana- the only studies were carried out in some entities of Lepi- lysed for the first time. In the Asteraceae this feature, daploa complex (Robinson, 1987a, 1987b, 1988a, 1988b, along with the morphology of style, has been very impor- 1990) and the most recently, in the genus Lessingianthus, tant taxonomically (Keeley & Robinson, 2009). However which also lists and describes the morphology of the tri- there are no studies using this character at species or chomes (Angulo & Dematteis, 2014). genus level among American Vernonieae. In Chryso- The results of this study show that all species of Chrys- laena, the presence or absence of glandular trichomes olaena are homogeneous in the presence of stigmatic type IIIA on branches style varies between species hence papillae in corolla lobes. In and Lessingian- was useful to distinguish some taxa. Most species have thus this feature varies with species with papillae and spe- this type of trichome in branching style, except C. guar- cies lacking these (Angulo & Dematteis, 2014; Isawumi, anitica, C. obovata, and C. simplex. 1999). Excluding the presence of papillae, the corolla pubescence was the most variable character and provided greater taxonomic information. In the florets of the species Basal stylar node analysed the five morphological types of trichomes The presence of basal stylar node varies greatly among described (I, II, IIIA, IIIB, and IV) are present in different taxa of the same species and among populations. This var- combinations. However, some entities lack glands in iation was also observed in Lessingianthus. Species such corolla lobes, such as C. simplex, C. guaranitica, and two as L. brevifolius (Less.) H. Rob., L. carvalhoi (H. Rob.) populations of C. obovata, which exhibit only non-glan- H. Rob., L. durus (Mart. ex DC.) H. Rob., L. regis (H. dular trichomes type I. This distinctive feature distin- Rob.) H. Rob., and L. santosii H. Rob. present a slight guishes them from other species. The exclusive presence development of the stylar node with several rows of thick- of glandular trichomes on corolla lobes was observed in walled cells (Angulo & Dematteis, 2014) as noted in some species of the genus Lessingianthus (Angulo & some of the entities of Chrysolaena. Dematteis, 2014). Additionally, C. nicolackii and C. sim- The basal stylar node was a character used for the clas- plex differ from other species of the genus by trichomes sification of Vernonieae of both hemispheres. It is consid- type IIIB and IV, respectively, on the corolla tube. The ered typical in many members of Neotropical Vernonieae presence of trichomes on the corolla tube was also and characteristic of some genera of the Lepidaploa com- reported in some species of Lessingianthus (Angulo & plex (Robinson, 1988a). The genus Lepidaploa presents a Dematteis, 2014) and Baccharoides (Isawumi et al., well-developed basal node. However, in three genera of 1996), but they differ morphologically from trichomes of the complex (Chrysolaena, Stenocephalum Sch. Bip., and Chrysolaena. Lessingianthus) the node seems lost or poorly differenti- This study noted for the first time the presence of twin ated (Robinson, 1988a, 1988b, 1990). It has been sug- hairs on the corolla of Chrysolaena (Vernonieae). As pre- gested that the lack of node is due to a loss. In viously mentioned, traditionally such trichomes are con- Vernonieae, apparently the basal node does not play a sig- sidered typical and unique to Asteraceae cypselae (Hess, nificant role, so there is no variability between groups 1938; Ramayya, 1962; Robinson, 2009; Roth, 1977). and, therefore, the observed changes in complex Lepida- However, it has been observed that the morphology and

Downloaded by [181.1.176.133] at 07:22 30 January 2016 ploa members should be considered rare and random ontogeny of trichomes of the corollas of some Onoseris (Robinson, 1990). From the taxonomic point of view, Willd., Cass. and Uechtritzia Freyn (Muti- although its absence is considered typical of Chrysolaena, sieae) species are the same as the twin hairs of cypselae. apparently, this character is not well defined in the genus Despite having a great morphological variation, all show (also in Lessingianthus) and, therefore, does not have the same ontogeny (Sancho & Katinas, 2002). Based upon much systematic value. this background in other Asteraceae groups, the trichomes type II observed in this study in corollas of Chrysolaena correspond to the morphology of a ’twin hair’. Neverthe- less, ontogenetic studies should be performed to confirm Base of anther thecae and apical appendage their origin. Chrysolaena has calcarate base of anther thecae, as well as Lessingianthus and Lepidaploa (Angulo & Dematteis, 2014). The shape can be sagittate or cleft. As in most of the Asteraceae, all Chrysolaena species Style have an apical anther appendage. This structure can be The general morphology of style coincides with most Ver- seen as an adaptation to protect the pollen from humidity nonieae and it was not a useful taxonomic character, since and insect predators, until the stigma and style push it up all species had the same characteristics. for the presentation of pollen (Stuessy, Sang, & Devore, 238 G. M. Via do Pico et al.

1996). The apical appendages are important among the However, C. guaranitica, C. simplex, and two populations New World Vernonieae, since variations occur in the of C. obovata do not have glandular trichomes, a typical presence of glands or thickening. However, the Old World characteristic of the fruits of Lessingianthus and most taxa are very uniform (Keeley & Robinson, 2009). Most Lepidaploa species (Dematteis 2007, 2009; Robinson entities of Chrysolaena have an ovate apical appendage. 1988a, 1988b, 1990, 1999). However, other forms (ovate-lanceolate, lanceolate, and The structure of pappus of Chrysolaena species is orbicular) were also observed. This character allowed sep- common among Lepidaploinae taxa (Vernonieae), and aration C. simplex and C. guaranitica from the other spe- also in other tribes such as Eupatorieae (Ageratina cies, since they were the only taxa with orbicular apical Spach) and Astereae (Symphyotrichum Nees) (Robinson, appendages. 2009). Regarding pubescence, Robinson (1988b, 1990) states King & Robinson (1966) have shown that special that numerous small glands are present in apical anther features of the carpopodium can be useful taxonomic appendages of Vernonieae and this characteristic is typi- characters. Many genera of Asteraceae could be differ- cal of Chrysolaena. Dematteis (2007) used this character, entiated on the basis of shape and cellular structure of in combination with pollen morphology (type ’C’) and the carpopodium (King & Robinson, 1966, 1987). base chromosome number (x D 10), for transferring to Haque and Godward (1984) examined the carpopodium two species, Lessingianthus simplex (Less.) H. Rob. and and its usefulness in the taxonomy of the Asteraceae. L. desertorum (Mart. ex DC.) to Chrysolaena (previously AccordingtoRobinson(2009), in general, the Verno- located in Lessingianthus subgen. Oligocephalus), nieae has a carpopodium of uniform characteristics, because they have glandular apical appendages and cypse- except Camchaya Gagnep. that lack a carpopodium. lae. However, this study demonstrates that C. simplex can Previous studies in some species of Chrysolaena have present an apical appendage without glands, a typical described a turbinate carpopodium formed by oblong characteristic of Lessingianthus and most species of Lepi- cells (Robinson, 1988b). The results of this study show daploa. Despite being closely related genera, a distin- that all species of Chrysolaena have a carpopodium guishing feature between C. simplex and Lessingianthus is with uniform characteristics: differentiated in the the shape of the apical appendages. In Lessingianthus the cypsela base, cylindrical- turbinate and formed by sub- appendages are ovate, ovate-lanceolate, lanceolate, or lin- square or slightly oblong cells. ear (Angulo & Dematteis, 2014), and this study has shown that C. simplex has orbicular apical appendages. The absence of glands in anthers was also denoted in previous Idioblasts studies as uncommon in C. obovata (sub nom. D Chryso- In plant histology, idioblasts are isolated cells that differ laena herbacea) (Robinson, 1988b) and in C. guaranitica in shape, size, content, and structure of the wall elements (Dematteis, 2007). with respect to surrounding tissue (Foster, 1955). It is a term with more than a specific morphological or physio- logical conventional connotation use, because it applies to Cypsela, pappus, and carpopodium a variety of cell types (Foster, 1955).

Downloaded by [181.1.176.133] at 07:22 30 January 2016 Isawumi (1996, 1999) and Robinson (2009) refer to idi- Wagenitz (1976) suggested that morphoanatomical stud- oblasts as the structure commonly found on the cypselae ies of the fruits of the Asteraceae might aid in elucidating walls of numerous Vernonieae. These are arranged in its systematics. Characteristics of the indumentum (Ritter groups of three or more cells and scattered or in longitudi- & Miotto, 2006), pappus, carpopodium (Haque & God- nal rows. There is no background on the structure or func- ward, 1984), and the anatomy of the pericarp (Bruhl & tion of these elements within the group, but the presence Quinn, 1990), or a combination of all of the characters or absence of idioblasts has been used as a taxonomic cited above have been used to delimit tribes, genera, and even species of this family. According to Bremer (1987), character (Isawumi et al., 1996; Robinson, 2009). These structures were observed in the fruits of some species of cypselae and pappus provide a wealth of characters at the Chrysolaena analysed in this study. However, their pres- lower taxonomic levels because of their considerable ence was highly variable among species and populations variation. of the same species, so it was not a useful basis for sys- The results of this study show that the shape and pubes- tematics of the group. cence of cypselae, as well as pappus features are fairly uniform among Chrysolaena species. All entities analysed have cypselae with pappus, non-glandular trichomes type Ovary wall crystals II (twin hairs), and most also glandular trichomes type Calcium oxalate (CaOx) crystals are found in over 215 IIIA. Both types of trichomes are biseriate and occur in plant families and distributed in organs such as stems, most genera of the Asteraceae family (Robinson, 2009). roots, leaves, floral structures, and seeds (Franceschi & Floral microcharacters of Chrysolaena (Asteraceae) 239

Horner, 1980; Lersten & Horner, 2000; Prychid & Rudall, identify the same groups of taxa with common characteris- 1999). Although their functional significance in plant tics (Figs 66 and 67). For both analyses it can be deduced development remains unclear, various functions have that the shape of apical anther appendages and the pres- been attributed to them, including calcium regulation in ence/absence of certain types of trichomes on certain struc- plant cells (Franceschi, 1989), protection against herbiv- tures are the determining variables in taxa grouping. ory (Molano-Flores, 2001), detoxification of heavy metals The statistical analyses clearly showed that C. simplex or oxalic acid (Franceschi & Nakata, 2005), tissue and C. guaranitica (Group 1) are very similar to each strength, light gathering, and reflection (Franceschi & other by the presence of orbicular apical appendage and Horner, 1980; Kuo-Huang, Ku, & Franceschi, 2007). the absence of glands in the lobes of the corolla, apical Even though the size, location, and other properties of the appendages, and cypselae. In turn, these characteristics crystals in may be affected by physical, chemical are very different from other species Chrysolaena, and and biological conditions, it is thought that crystal forma- grouped separately from them. With respect to the remain- tion within the cell is under genetic control (Ilarslan, ing species (Group 2), one can identify remarkably consis- Palmer, & Horner, 2001). Thus the type, presence or tent subgroups based on common features that are mainly absence of crystals may be represented as a taxonomic the presence/absence of certain types of trichomes on flor- character (Lersten & Horner, 2000; Prychid & Rudall, ets structures. However, in a significant number of taxa 1999). there is overlap of characters and separation of species in Ovary wall crystals have been successfully studied the innermost clusters becomes difficult. Populations of for the taxonomic evaluation of several tribes in the different species had shared characters, which is reflected family Asteraceae (Dormer, 1961;Isawumi,1995a, in the polyphyletic terminals in the phenogram (UPGMA) 1995b;Isawumietal.,1996; Nordenstam & El-Gha- consisting of taxa from different species and the separa- zaly, 1977). Studies by Robinson (1988a, 1988b, 1999) tion of populations of the same species in different clus- suggest that the elongated crystals (the correct term ters. This is seen equivalently in the scatterplot (PCoA) would prismatics, because it is a structure with vol- and taxa of different species with the same coordinates ume) of the fruit wall are typical of the sorts Chryso- and populations of the same species with different laena and Lepidaploa; opposed to Lessingianthus, coordinates. which is generally square crystals sub squares (cubic or nearly cubic). However, in this study four types of crystals were found on the cypselae of Chrysolaena: druses, styloid, prismatic, and cubic. Other studies Taxonomic implications have also observed druses, styloid, and prismatic crys- Table 3 shows and compares the microcharacters of Chrys- tals in the ovary walls and carpopodium of C. platensis olaena, Lepidaploa,andLessingianthus based on the results [sub nom. Vernonia platensis] (Galastri & Oliveira, of this study and previous information from the literature. 2010)andC. obovata [sub nom. V. herbacea](Martins As previous studies indicated, Chrysolaena, Lepida- & Oliveira, 2007). Besides presenting various forms, ploa, and Lessingianthus share some characters (Robin- the presence of different types of crystals in Chryso- son, 1988a, 1988b). The results of this study agree with laena varied to interspecific and intraspecific level. this background. Although most Chrysolaena species are Downloaded by [181.1.176.133] at 07:22 30 January 2016 From the taxonomic point of view, the results of this homogeneous in the typical microcharacters of the genus, analysis suggest that while the prismatic crystals some entities may have typical characteristics of Lessin- (elongated in terms of Robinson, 1988b) are among the gianthus (such as apical anther appendages and cypselae most common forms observed in Chrysolaena species, without glands) or Lepidaploa (style with basal node). they do not constitute a distinctive character of the genus Chrysolaena simplex, C. guaranitica and some popula- because various shape crystals were observed in most tions of C. obovata show systematic inconsistencies since entities. Besides, the morphology and the presence of they have microcharacters that show a departure from crystals are not constant between Chrysolaena species, so Chrysolaena and are closer to Lessingianthus. However, this would not be a useful character to recognize species previous studies have reported base chromosome number taxonomically. x D 10 in C. simplex and C. obovata (Ruas, Ruas, Vieira, Matzenbacher, & Martins, 1991; Via do Pico & Demat- teis, 2012), presence of glands on corolla lobes, anther appendages and cypselae in C. guaranitica and C. simplex Statistical analyses: UPGMA and PCoA (Dematteis, 2007, 2009), and pollen grains type ’C’ in all The results of the statistical analyses performed (UPGMA these species (Via do Pico & Dematteis, 2013a), which sup- and PCoA) correspond to each other. The grouping of spe- ports the taxonomic position of these entities within Chryso- cies according to the analyses of clusters is consistent with laena. Lessingianthus always has basic chromosome the clustering seen in the diagram PCoA. Both graphs number x D 16 and type ’B’ pollen, while Lepidaploa 240 G. M. Via do Pico et al.

Table 3. Microcharacters of Chrysolaena, Lessingianthus and Lepidaploa based on previous studies and the results of this study (Angulo & Dematteis, 2014; Dematteis 2007, 2009; Robinson 1988a, 1988b, 1990, 1999).

Microcharacters Chrysolaena Lepidaploa Lessingianthus

Trichomes of corolla lobes Glandular and non-glandular, rarely Glandular Glandular and non-glandular only non-glandular Apical anther appendages Glandular, rarely non-glandular Rarely glandular Non-glandular Basal stylar node Present or absent Present Rarely present Cypselae Glandular, rarely non-glandular Non-glandular, rarely glandular Non-glandular Ovary wall crystals Cubic, prismatic, estiloid, druses Prismatic Cubic

presents x D 14, 15 or 16 and pollen grains ’C’, ’D’ or ’G’ type IIIB on the corolla tube and C. dusenii may have tri- (Angulo & Dematteis, 2010; Dematteis, 2009). chomes type I on corolla lobes. According to Robinson (1988b), although Chrysolaena Another group of related species is C. guaranitica, C. violates the generic boundaries between Lessingianthus desertorum, and C. simplex. Chrysolaena guaranitica and and Lepidaploa in some secondary characters, the pres- C. desertorum differ from C. simplex by monocephalic ence of glands on the apical anther appendage is the only inflorescence; and C. desertorum differs from C. guarani- microcharacter that allows separation of the genus from tica by glabrous corolla lobes (Dematteis, 2009). How- the other members of the complex Lepidaploa. Although ever, in this study we observed specimens of C. some species lack glands, their presence is too consistent desertorum with glabrous corolla and glandular and/or in the genus to be ignored. In this sense, the results non-glandular trichomes (type I or II). Besides, C. guar- obtained here support the points made by the author. The anitica and C. simplex are distinguished from C. deserto- presence of apical anther appendage and glandular cypse- rum by the absence of glands on corolla lobes (it has non- lae are common characteristics among species Chryso- glandular trichomes type I and occasionally type II), on laena. In addition to these, this study shows that presence style, cypselae, and the orbicular shape of apical anther of glands on the style and corollas is another consistent appendage. The trichomes type IV on the corolla tube dis- characteristic in the genus. However, the absence of basal tinguishes C. simplex from C. guaranitica. stylar node would not be a diagnostic character since this By the arrangement of leaves in a basal rosette, C. oligo- varies widely among species. phylla, C. propinqua,andC. flexuosa are related species. Regarding ovary wall crystals, contrary to what is However, C. oligophylla differs from these two entities by stated in previous studies (Robinson, 1988b), Chrysolaena the pedunculate heads and the umbeliform or pseudo- may have more than one type of crystal (cubic, prismatic, umbeliform inflorescence; while C. propinqua differs from styloid, and druses), and this allows differentiation from C. flexuosa by cylindrical or narrowly campanulate involu- Lessingianthus and Lepidaploa which have one type of cre (Dematteis, 2009). The different combinations of tri- crystal (Dematteis, 2009). chomes on floral structures allow differentiation. Downloaded by [181.1.176.133] at 07:22 30 January 2016 At species level, the results show that Chrysolaena spe- Chrysolaena campestris and C. lithospermifolia are cies differ from each other by having different combina- similar. Both entities are mainly differentiated by the tions of microcharacters. The genus presents some species bracts size of the inflorescence and the leaf length closely similar morphologically and these micro-morpho- (Dematteis, 2009). Chrysolaena campestris is distinguish- logical differences are useful to differentiate them. Chryso- able by the presence of non-glandular trichomes on laena cristobaliana, C. cognata, C. sceptrum, C. platensis, corolla lobes and apical anther appendages in addition to and C. cordifolia, for example, are a closely related group. glands. Based on their morphological similarity, C. cande- The results of this study shows that they can be differenti- labrum and C. verbascifolia have been considered as syn- ated by the shape of the apical anther appendages, the tri- onyms (Zuloaga, Morrone, Belgrano, Marticorena, & chome types on corolla lobes, the presence of basal node Marchesi, 2008). However, they differ by the indumentum and the cypsela shape (see Tables 1 and 2). type and phyllaries shape (Dematteis, 2009). Micro-mor- Chrysolaena dusenii and C. nicolackii are very similar phologically, C. verbascifolia can also be distinguished in morphology and geographical distribution. Micro-mor- from C. candelabrum by the type IIIB trichomes on phologically they can only be differentiated by the florets corolla lobes and turbinate cypselae. Chrysolaena cande- number of capitula and the leaves being either discolorous labrum is distinguished by the four types of calcium oxa- (C. dusenii) or concolorous (C. nicolackii) (Dematteis, late crystals on the ovary walls. 2009). Micro-morphologically they differ from each other Chrysolaena obovata has some resemblance to C. cris- by the fruit shape and because C. nicolackii has trichomes tobaliana and C. sceptrum, from which it differs by ovate Floral microcharacters of Chrysolaena (Asteraceae) 241

leaves and the florets number per capitula (Dematteis, taxonomic implications. Plant Systematics and Evolution, 2009). From the point of view of microcharacters it differs 300, 1925À1940. from these two entities because its style, anther appen- Appezzato-da-Gloria, B., Da Costa, F. B., da Silva, V. C., Gobbo-Neto, L., Garcia Rehder, V. L., & Hayashi, A. H. dages and cypselae are non-glandular and its cypselae are (2012). Glandular trichomes on aerial and underground cylindrical-turbinate. organs in Chrysolaena species (VernonieaeÀAsteraceae): Structure, ultrastructure and chemical composition. Flora, 207, 878À889. Conclusions Appezzato-da-Gloria, B., Hayashi, A. H., Cury, G., Soares, M. K. M., & Rochao, R. (2008). Occurrence of secretory struc- This study analysed for the first time the floral microchar- tures in underground systems of seven Asteraceae species. acters of genus Chrysolaena. Microcharacters previously Botanical Journal of Linnean Society, 157, 789À796. studied were considered, and also new characters were Bremer, K. (1987). Tribal interrelationships of the Asteraceae. Cladistics, 3, 210À253. revealed, which contributed taxonomically. The presence/ Bruhl, J. J., & Quinn, C. J. (1990). Cypsela anatomy in the absence and types of trichomes on different floral and fruit ‘Cotuleae’ (Asteraceae-Anthemideae). Botanical Journal of structures (corolla, style and anthers), and also the shape the Linnean Society, 102,37À59. of the apical anther appendage were the most informative. Carlquist, S. (1958). Structure and Ontogeny of Glandular Tri- Besides, the types of trichomes of the floral structures and chomes of Madinae (Compositae). American Journal of Bot- any, 45, 675À682. cypselae were described and typified for the first time. Carlquist, S. (1961). Comparative plant anatomy. New York: The results indicate that most of the floral microchar- Holt, Rinehart, and Winston. acters of Chrysolaena analysed are quite consistent in the Dematteis, M. (2007). Taxonomic notes on the genus Chryso- genus. However, until now, the pollen morphology and laena (Vernonieae, Asteraceae), including a new species À the chromosome base number are most useful for separat- endemic to Paraguay. Annales Botanici Fennici, 44,56 64. Dematteis, M. (2009). Revision taxonomica del genero sudamer- ing Chrysolaena from the related genera Lessingianthus icano Chrysolaena (Vernonieae, Asteraceae). Boletın de la and Lepidaploa. Sociedad Argentina de Botanica , 44, 103À170. At species level, the results show that related species Di Rienzo, J. A., Casanoves, F., Balzarini, M. G., Gonzalez, L., can be discriminated by the different combinations of Tablada, M., & Robledo, C. W. (2012). InfoStat, version À microcharacters. The value of floral microcharacters as 2012. Argentina: Grupo InfoStat, FCA UNC. Dormer, K. J. (1961). The crystals in the ovaries of certain Com- taxonomic criterion could be increased if combined with positae. Annals of Botany, 25, 241À254. other lines of evidence such as pollen morphology and Ekman, E. L. (1914). West Indian Vernonieae. Arkiv for€ Botanik, chromosome number. 13,1À106. Faust, W. Z. (1972). A biosystematic study of the Interiores spe- cies group of the genus Vernonia (Compositae). Brittonia, 24, 363À378. Acknowledgements Faust, W. Z., & Jones, S. B. (1973). The systematic value of tri- This work was supported by grants from the Consejo chome complements in a North American group of Vernonia Nacional de Investigaciones Cientıficas y Tecnicas (Compositae). Rhodora, 75, 517À528. (CONICET). Foster, A. S. (1955). Plant idioblast: Remarkable examples of cell specialization. Protoplasma, 46, 184À193. Franceschi, V. R. (1989). Calcium oxalate formation is a rapid

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